We have successfully assembled a full-length infectious construct of Mouse Hepatitis Virus (MHV-A59), a group II coronavirus. MHV is the prototype for studying the mechanisms of coronavirus replication, transcription, replicase protein function, pathogenesis, cross species transmission, and assembly. This proposal focuses on the function of the MHV ORF1a c-terminal replicase proteins in RNA transcription and replication, and on the molecular mechanisms governing MHV cross species transmission and replication efficiency in alternative hosts. In aim 1, we test the hypothesis that the MHV p10, p22, p12 and p15 (p10-p15) replicase proteins function in viral transcription and replication. We will map functional domains in these proteins by systematically deleting or duplicating sites in each of the small replicase proteins. Rescue of selected lethal mutations will be tested with the appropriate MHV replicase proteins and precursors encoded in trans using alphavirus replicons or other expression vectors. The effect of these mutations/deletions on virus replication, replicase protein expression and processing, membrane localization, replication complex formation and RNA synthesis will be evaluated using standard techniques. In aim 2, the p15 replicase protein will be systematically mutated using cluster charged amino acid to alanine mutagenesis and site specific mutagenesis in selected sites of predicted structure and function. The goal is to isolate a spectrum of mutants with informative phenotypes that map functional domains in the p15 replicase protein. In aim 3, we hypothesize that at least two distinct evolutionary pathways contribute to MHV cross species transmission. We will use reverse genetics approaches to identify the specific alleles in the S glycoprotein that mediate cross species transmission and altered MHV receptor usage. We will determine if mutations in HE and/or the replicase also contribute to MHV-MCF7 and MHV-H2 replication fitness in human and hamster cells, respectively. These experiments will particularly enhance our overall understanding of coronavirus host range expansion, virus-receptor interactions, and replicase protein processing and function in RNA transcription and host range replication efficiency.